It is well known that nucleic acids, i.e., deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) are essential building blocks in living cells. These compounds are high molecular weight polymers which are made up of many "nucleotide" units, each such nucleotide unit being composed of a base (a purine or a pyrimidine), a sugar (which is either ribose or deoxyribose) and a molecule of phosphoric acid. DNA contains deoxyribose as the sugar moiety and the bases adenine, guanine, cytosine, and thymine (which may be represented as A, G, C and T, respectively). RNA contains ribose instead of deoxyribose and uracil (U) in place of thymine.
The nucleotide units in DNA and RNA are assembled in definite linear sequences which determine specific biological functions. In the normal mammalian cell, DNA replicates itself, and also serves as the template for the synthesis of RNA molecules whose nucleotide sequences carry the information encoded by the DNA. RNA molecules serve several different functions within the cell. Messenger RNA (mRNA) directs protein synthesis.
According to the well known Watson-Crick model, DNA molecules consist of two polynucleotide strands coiled about a common axis. The resulting double helix is held together by hydrogen bonds between complementary base pairs in each strand. Hydrogen bonds are formed only between adenine (A) and thymine (T) and between guanine (G) and cytosine (C). Hence within the double helix, adenine (A) and thymine (T) are viewed as complementary bases which bind to each other as A-T. The same is true for guanine (G) and cytosine (C), as G-C.
In a single polynucleotide strand, any sequence of nucleotides is possible. However, once the order of bases within one strand of a DNA molecule is specified, the exact sequence of the other strand is simultaneously determined due to the indicated base pairing. Accordingly, each strand of a DNA molecule is the complement of the other. In the process of DNA replication, the two strands act as templates for the synthesis of two new chains with complementary nucleotide sequences. The net result is the production of two new DNA molecules each containing one of the original strands plus a newly synthesized strand that is complementary to it. This process is referred to as semiconservative replication and allows the genetic information encoded within the DNA, specified by the nucleotide sequence, to be transmitted from one generation to the next.
Collectively, the genetic information of an organism is termed the genome. The genome of bacteria and of all higher species is composed of DNA. The genome of viruses may be either DNA or RNA. In any case, the genome of any particular species, whether a virus, bacteria, or higher organism has a characteristic nucleotide sequence which, stated simply, can be viewed as the "fingerprint" of that species.
In the process of transcription, DNA is copied, or transcribed, into RNA. The RNA molecule that is synthesized is single stranded. Its nucleotide sequence is complementary to a segment of one of the two strands of the DNA molecule, and hence it is an exact copy of the opposite strand except that thymine is replaced with uracil. The length of DNA that is copied to form a single RNA molecule represents one gene. The human genome contains approximately 50,000 genes.
Messenger RNAs (mRNAs) carry the coding information necessary for protein synthesis. The sequence of nucleotides in the mRNA molecule specifies the arrangement of amino acids in the protein whose synthesis will be directed by that mRNA. Each set of three nucleotides, a unit called a codon, specifies a particular amino acid. The process by which mRNAs direct protein synthesis is termed translation. Translation of mRNAs occurs on ribosomes within the cytoplasm of the cell.
The flow of information from gene to protein can thus be represented by the following scheme: ##STR1##
For each protein made by an organism there exists a corresponding gene.
It is generally known in the field of molecular biology that oligodeoxyribonucleotides, short single-stranded DNA fragments, having a nucleotide sequence complementary to a portion of a specified mRNA may be used to block the expression of the corresponding gene. This occurs by hybridization (binding) of the oligonucleotide to the mRNA, according to the rules of base pairing described above, which then prevents translation of the mRNA. It has now been discovered that the inhibition of translation observed is due to cleavage of the mRNA by the enzyme RNaseH at the site of the RNA-DNA double helix formed with the oligonucleotide (see FIG. 1). Hybridization of the oligonucleotide to the mRNA is generally not sufficient in itself to significantly inhibit translation. Evidently an oligonucleotide bound to mRNA can be stripped off in the process of translation. An important corollary of this result is that for any modified oligonucleotide to efficiently block expression of a gene, the hybrid formed with the selected mRNA must be recognized by RNaseH and the mRNA cleaved by the enzyme.
The process of hybrid-arrested translation outlined in FIG. 1 has obvious implications for the use of oligonucleotides as therapeutic agents. By selectively blocking the expression of a particular gene essential for the replication of a certain virus or bacteria an oligonucleotide could serve as an antimicrobial agent. Similarly, an oligonucleotide targeted against a gene responsible or required for the uncontrolled proliferation of a cancer cell would be useful as an anticancer agent. There are also applications in the treatment of genetic diseases such as sickle cell disease and thalassemia for which no adequate treatment currently exists. Any gene can be targeted by this approach. The problem of drug specificity, a major hurdle in the development of conventional chemotherapeutic agents, is immediately solved by choosing the appropriate oligonucleotide sequence complementary to the selected mRNA. This circumvents toxicity resulting from a lack of selectivity of the drug, a serious limitation of all existing antiviral and anticancer agents.
In addition to the use of oligonucleotides to block the expression of selected genes for the treatment of diseases in man, other applications are also recognized. Additional applications include but are not limited to the use of such oligonucleotides in veterinary medicine, as pesticides or fungicides, and in industrial or agricultural processes in which it is desirable to inhibit the expression of a particular gene by an organism utilized in that process.
A major problem limiting the utility of oligonucleotides as therapeutic agents is the rapid degradation of the oligonucleotide in blood and within cells. Enzymes which degrade DNA or RNA are termed nucleases. Such enzymes hydrolyze the phosphodiester bonds joining the nucleotides within a DNA or RNA chain, thereby cleaving the molecule into smaller fragments.
In the past there has been some progress made in the development of oligonucleotide analogs that are resistant to nuclease degradation, but the use of such derivatives to block the expression of specifically targeted genes has met with limited success. See, for example, Ts'o et al. U.S. Pat. No. 4,469,863 issued Sep. 4, 1984; Miller et al. U.S. Pat. No. 4,507,433 issued Mar. 26, 1985; and Miller et al. U.S. Pat. No. 4,511,713 issued Apr. 16, 1985. Each of the above patents have in common the objective of blocking the expression of selected genes, but the approach taken has been less than successful judging from the high concentrations of oligonucleotide required and a corresponding lack of selectivity. The work described in each of the three prior patents mentioned involves the use of oligonucleotides in which all of the phosphate groups have been modified in the form of methylphosphonates: ##STR2## B, and B.sub.2 are nucleic acid bases (either A, G, C or T).
In particular, U.S. Pat. No. 4,469,863 involves inter alia, methylphosphonate modification of oligonucleotides. U.S. Pat. No. 4,507,433 involves a process for synthesizing deoxyribonucleotide methylphosphonates on polystyrene supports; and U.S. Pat. No. 4,511,713 involves determining the base sequence of a nucleic acid and hybridizing to it an appropriately synthesized oligonucleotide methylphosphonate to interfere with its function.
The low level of activity observed using these fully modified methylphosphonate analogs led us to suspect that they would not form effective substrates for RNaseH when hybridized with mRNAs. Results presented in Example 2 show this to be the case.
In an effort to design new oligonucleotide analogs of greater use as therapeutic agents, we undertook a study of the pathways by which oligonucleotides are degraded within blood and within cells. We have discovered that the sole pathway of degradation in blood (Example 3) and the predominant pathway in cells (Example 4) is via sequential degradation from the 3'-end to the 5'-end of the DNA chain in which one nucleotide is removed at a time. This pathway of degradation is illustrated in FIG. 2. These results provided for the first time a rational basis for the design of oligonucleotide analogs modified so as to inhibit their degradation without interfering with their ability to form substrates for RNaseH.
It has now been discovered that oligonucleotides modified at only the 3'-most internucleotide link are markedly protected from degradation within blood and within cells (Examples 5 and 6). Moreover, we have found that such derivatives have normal hybridization properties and do form substrates with mRNAs that are recognized and cleaved by RNaseH, thereby preventing expression of the targeted gene (Example 7).
The accomplishment of the inhibition of expression of selected genes by oligonucleotides that are resistant to degradation, and that are, therefore, more effective when used therapeutically, is the primary objective of this invention.
Other objectives of the present invention include the preparation of oligonucleotides modified at the 3'-phosphodiester linkage and the formulation of the modified oligonucleotide in a manner suitable for therapeutic use. It is also apparent that DNA or RNA molecules so modified would be useful as hybridization probes for diagnostic applications, in which case, the modification of the 3'-internucleotide link would inhibit degradation of the probe by nucleases which may be present within the test sample.